Endocrine Abstracts

The gonadotrophin receptors, luteinising hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) are G-protein coupled receptors, vital in regulating reproductive functions. Whilst FSHR and LHR are known to form homomers, and have been recently shown to heterodimerise/oligomerise, the functional and physiological significance of LHR/FSHR heteromerisation remains elusive. This study aimed to explore the functional significance of LHR/FSHR crosstalk, exploring mechanistic detail in HEK293 cells, and translating findings into primary human ovarian granulosa cells. Using HEK293 stably expressing LHR +/− transient-transfection of FSHR, the pattern of LH-induced increase in intracellular calcium signalling, measured via Fluo-4direct indicator dye and confocal imaging, was significantly altered in the presence of FSHR, from an acute and rapid signal to a sustained calcium response. To ascertain the molecular mechanisms governing this change, pharmacological inhibitors were employed. Inhibition of Gαq/11 with UBO-QIC and Gαi with pertussis toxin showed the full calcium signal and profile was Gαq/11 dependent and Gαi independent. Interestingly, the sustained LH-dependent calcium profile was nifedipine-sensitive, indicating that crosstalk between LHR and FSHR enabled LH-dependent activation of L-type calcium channels. Moreover, inhibition of βγ subunits and PI3Kinase activation with gallein and wortmannin respectively, resulted in a sustained calcium signalling profile in cells expressing LHR alone, but had no further effect on LHR/FSHR calcium profile, suggesting FSHR alters the ability of an LHR-Gβγ-PI3Kinase pathway to inhibit L-type calcium channel activity. Translation of this work to primary human granulosa lutein cells, that co-express the LHR and FSHR, revealed an intact LH-dependent calcium response that was nifedipine-sensitive, indicating that LH-dependent activation of L-type calcium channels is an integral pathway with potential physiological significance in vivo. Together these data suggest that crosstalk between LHR/FSHR may represent a key mechanism for generating sustained LH-mediated signal responses in the ovary, responding to the changing physiological requirements of LHR signalling.